Detection of HPV

ABSTRACT

The present invention provides compositions and methods for the detection and characterization of HPV sequences. More particularly, the present invention provides compositions, methods and kits for using invasive cleavage structure assays (e.g. the INVADER assay) to screen nucleic acid samples, e.g., from patients, for the presence of any one of a collection of HPV sequences. The present invention also provides compositions, methods and kits for screening sets of HPV sequences in a single reaction container.

The present application is a divisional of U.S. patent application Ser.No. 11/857,942, filed Sep. 19, 2007, which is a continuation of U.S.patent application Ser. No. 10/951,241, filed Sep. 27, 2004, whichclaims priority to U.S. Provisional Application Ser. No. 60/505,786,filed Sep. 25, 2003, each of which is herein incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention provides methods and composition related tonucleic acid detection assays for use in basic research, clinicalresearch, and for the development of clinical detection assays. Inparticular, the present invention provides methods for characterizinghuman papillomavirus (HPV) sequences.

BACKGROUND

Cervical cancer accounts for nearly 10% of all female cancers and is aleading cause of cancer among women in developing countries (Franco, E.L. et al., Can Med Assoc J. 2001; 164:1017-25). The regions with thehighest incidence of the disease are generally those with the greatestmortality and include Central America, Africa, and the Carribean(Ferlay, J. et al., 1998. IARC CancerBase no. 3. Lyon:IARCPress.).Incidence in Europe and North America has declined precipitously overthe past 50 years, possibly due to the advent of routine screening byPapanicolaou (Pap) smear testing (reviewed in Franco et al., ibid).Cervical cancer is one of the most preventable cancers, with survivalbeing directly related to the stage of the disease at diagnosis. The5-year survival rate is 88% for women having initial diagnosis oflocalized disease as opposed to 13% for women diagnosed with distantdisease (Report of the Gynecologic Cancers Progress Review Group,November 2001, National Cancer Institute). More than 50% of womendiagnosed with cervical cancer in the U.S. have not had a Pap smear inthe past three years (Wright, T. C. et al., JAMA. 2000; 283:81-6).

Pap screening remains the predominant mode of detecting cancerous andprecancerous cervical lesions; more than 50 million women undergo Papscreening each year in the U.S. (Wright, T. C. et al., JAMA 2002;287:2120-29). Despite its widespread use, Pap smear testing is onlypartially effective; some estimates place the sensitivity ofconventional Pap smear testing at 50-60% (Lorincz, A. T. and Richart, R.M., (Arch Pathol Lab Med. 2003; 127:959-68; Nanda, K. et al., 2000. AnnIntern Med 132:810.; Fahey M T, et al. Am J. Epidemiol. 1995; 141:680-9;Myers E R, McCrory D C, Subramanian S, et al. Obstet. Gynecol. 2000;96:645-52.) or 70-80% (Clavel, C. et al., 2001. Br J Cancer 84:1616).Recent innovations in cytological screening and sampling, such asliquid-based tests, have improved the sensitivity of these methods to75-95% (Lorincz, A. T. et al. ibid; Nanda, K. et al., ibid.; HutchinsonM L, Zahniser D J, Sherman M E, et al. Cancer. 1999; 87:48-55.).Nonetheless, even these improved methods fail to detect a significantportion of abnormal, and often precancerous, cells. Once identified,patients with atypical squamous cells of undetermined significance(ASCUS) are subjected to various levels of monitoring and treatment,depending on the particular attendant risk factors and clinicalpresentation (reviewed in Wright, T. C. et al. JAMA 2002, ibid).

Human Papillomavirus (HPV) has been identified as the primary, andpossibly only, cause of cervical cancer (Muñoz N, Bosch F X, deSanjoséS, et al., Int J Cancer 1992; 52:743-9; Bosch F X, Lorincz A,Munoz N, Meijer Shah K V., Clin Pathol 2002; 55:244-65), implicated inas many as 99.7% of all cases (Wallboomers, J. M. et al., 1999. J Pathol189:12-19). The HPV genome is an 8 kb, circular, double stranded DNAcomprising 8 genes, all encoded on the same strand. As many as 200different HPV types have been identified in humans (Burd, E. M. ClinMicrobiol Rev. 2003; 16:1-17); of these approximately 40 types have beenfound capable of infecting the genital tract (Munoz, N. N Engl J Med2003; 348:518-27.). Still further classification has resulted in theidentification of high- and low-risk viral types for development ofcervical cancer. Estimates place the number of high-risk types between13-19 strains, with two strains, HPV 16 and 18 together accounting foras much as 55-85% of infections, depending on subject age andgeographical location (Munoz, N., ibid). The predominant low-riskstrains are HPV 6 and 11; these may lead to genital warts (reviewed inBurd, E. M., ibid).

The elucidation of certain high risk HPV strains as the causative agentsof cervical cancer, coupled with advances in molecular biologicalmethods, has expanded the spectrum of methods available for bothpreventing and detecting HPV infection. Vaccines for the most commonhigh-risk HPV strains are currently in clinical trials (Koutsky, L A. etal., 2002. NEJM 347:1645-51). Moreover, some authorities are calling forHPV DNA screening for use in conjunction with, or in some cases, in lieuof, conventional cytological methods (Wright, T. C. and Schiffman, M. N.Engl. J. Med, 2003; 348: 489-90). Various alternative DNA-baseddetection methods have been introduced, including the HYBRID CAPTURE II(HCII) test (Digene, Gaithersburg, Md.), which was been approved by theFDA in March, 1999. The HYBRID CAPTURE method relies on hybridization oftarget DNA to complementary RNA probes. The resultant RNA-DNA hybridsare recognized by surface-bound antibodies as well as antibodiesconjugated to alkaline phosphatase, allowing generation of achemiluminescent signal in the presence of appropriate substrates(Lorincz, A. T. J Obstet Gynaecol Res. 1996; 22:629-36; also U.S. Pat.No. 4,908,306 and related patents and applications). Further alternativemethods include the use of sequence specific probes for use in PCR orsandwich hybridization assays, such as those described in U.S. Pat. No.6,583,278. Other methods rely on various PCR primers for selectiveamplification of specific strains, as in U.S. Pat. No. 5,447,839 andrelated applications. Still other methods rely on in situ hybridizationof sequence-specific probes to isolated cervical cells, described in WO00/24760A1 (e.g. INFORM HPV, Ventana Medical Systems, Inc., Tuscon,Ariz.; Qureshi M N et al., Diagn. Cytopathol. 2003; 29:149-155).

Therefore, there exists a need for a rapid, sensitive, and highlyquantitative direct detection assay for detecting HPV infection by highrisk strains in cervical samples. Given the current reliance onmolecular methods, it is likely that there will be an ongoing andincreasing need for rapid, quantitative methods of detecting HPVinfection.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for thedetection and characterization of sequences associated with humanpapillomavirus (HPV). More particularly, the present invention providescompositions, methods and kits for using invasive cleavage structureassays (e.g. the INVADER assay, Third Wave Technologies, Madison, Wis.)to screen nucleic acid samples, e.g., from patients, for the presence ofany one or more of a collection of sequences associated with HPV. Thepresent invention also provides compositions, methods and kits forscreening sets of different HPV sequences in a single reactioncontainer. The present invention may be used to detect integrated and/ornon-integrated viral sequences.

In other embodiments, synthetic DNA suitable for use with the methodsand compositions of the present invention is made using a purifiedpolymerase on multiply-primed genomic DNA, as provided, e.g., in U.S.Pat. Nos. 6,291,187, and 6,323,009, and in PCT applications WO 01/88190and WO 02/00934, each herein incorporated by reference in theirentireties for all purposes. In these embodiments, amplification of DNAsuch as genomic DNA is accomplished using a DNA polymerase, such as thehighly processive Φ29 polymerase (as described, e.g., in U.S. Pat. Nos.5,198,543 and 5,001,050, each herein incorporated by reference in theirentireties for all purposes) in combination with exonuclease-resistantrandom primers, such as hexamers.

The method is not limited by the nature of the target nucleic acid. Insome embodiments, the target nucleic acid is single stranded or doublestranded DNA or RNA. In some embodiments, double stranded nucleic acidis rendered single stranded (e.g., by heat) prior to formation of thecleavage structure. In some embodiments, the source of target nucleicacid comprises a sample containing genomic DNA. Sample include, but arenot limited to, tissue sections, blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum and semen.

In some embodiments, the present invention provides methods of detectingan HPV sequence or method for diagnosing cancer, comprising; a)providing; i) a sample from a subject; and ii) a composition comprisingan oligonucleotide detection assay (e.g. as described herein); and b)contacting said sample with said composition such that the presence orabsence of at least one HPV sequence is determined. In some embodiments,the sample is a tissue section, blood sample, mouth swab sample, salivasample, or other biological fluid sample from the subject.

In some embodiments, the present invention provides a method fordetecting at least one HPV sequence in a sample, comprising using afirst and a second oligonucleotide, wherein the oligonucleotides areconfigured to form an invasive cleavage structure with a target sequencecomprising the at least one HPV sequence. In some embodiments, the firstoligonucleotide comprises a 5′ portion and a 3′ portion, wherein the 3′portion is configured to hybridize to the target sequence, and the 5′portion is configured to not hybridize to the target sequence. In someembodiments, the second oligonucleotide comprises a 5′ portion and a 3′portion, wherein the 5′ portion is configured to hybridize to the targetsequence, and wherein the 3′ portion is configured to not hybridize tothe target sequence. In preferred embodiments, the first and secondoligonucleotides are selected from the group consisting of SEQ ID NOS.1-5, 7-62, 64-67, 69-70, 73-116 and 122-193.

In some embodiments, the present invention provides a method fordetecting the presence or absence of HPV nucleic acid in a samplecomprising providing a sample comprising nucleic acids and an invasivecleavage assay configured to detect at least one HPV nucleic acid andexposing the sample to the detection assay under conditions such thatthe at least one HPV nucleic acid can be detected, and detecting thepresence or absence of HPV nucleic acid in a sample. In someembodiments, the detecting comprises identifying one or more strains ofHPV present in the sample. In preferred embodiments, the HPV strain isselected from the group consisting of, but not limited to, HPV 16,16Ty2, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 58 iso, 59, 66, 67, 68,68var, 69, 70, or 82. In some embodiments, the nucleic acid is amplifiedprior to said exposure step.

In some embodiments, the present invention provides a method fordetecting the presence or absence of HPV nucleic acid in a samplecomprising treating the sample using a first oligonucleotide and asecond oligonucleotide, wherein the oligonucleotides are configured toform an invasive cleavage reaction and detecting the presence or absenceof HPV nucleic acid. In particular embodiments, the oligonucleotidescomprise one or more oligonucleotides selected from the group consistingof, but not limited to, SEQ ID NOS. 1-5, 7-62, 64-67, 69-70, 73-116 and122-193. In some preferred embodiments, the oligonucleotidesindividually contain one or more mismatches with target HPV nucleicacid. In some embodiments, the oligonucleotides are configured tohybridize to non-HPV nucleic acid sequences or two hybridize to two ormore strains of HPV. In some embodiments, the oligonucleotides areconfigured such that a stable hybridization duplex between one or moreof the oligonucleotides and the HPV target nucleic acid is not formed.

In some embodiments, the target nucleic acid comprises genomic DNA ormRNA. In other embodiments, the target nucleic acid comprises syntheticDNA or RNA. In some preferred embodiments, synthetic DNA or RNA within asample is created using a purified polymerase. In some preferredembodiments, creation of synthetic DNA using a purified polymerasecomprises the use of PCR. In some preferred embodiments, creation ofsynthetic DNA comprises use of the methods and compositions foramplification using RNA-DNA composite primers (e.g., as disclosed inU.S. Pat. No. 6,251,639, herein incorporated by reference in itsentirety). In other preferred embodiments, creation of synthetic DNAusing a purified DNA polymerase suitable for use with the methods of thepresent invention comprises use of rolling circle amplification, (e.g.,as in U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, hereinincorporated by reference in their entireties). In other preferredembodiments, creation of synthetic DNA comprises amplification usingnucleic acids comprising loop-forming sequences, e.g., as described inU.S. Pat. No. 6,410,278, herein incorporated by reference in itsentirety.

In some embodiments, the present invention provides methods and kitsconfigured to detect more than one HPV strain in a single reactionvessel (e.g., kits and methods to detect all high risk strains in fouror fewer reactions). Thus, the present invention provides kits andmethods comprising pooled detection assay components. In some preferredembodiments, a single oligonucleotide in the pooled detection assaycomponents is configured to take part in an invasive cleavage structurein the presence of two or more HPV target strains. The pooled detectionassay components also find use in methods and kits using detectiontechnologies other than invasive cleavage technology. For example, thepooled detection assays for detection of HPV sequences (e.g., whereinone or more oligonucleotides find use in detecting multiple HPVsequences in a single reaction) provided in the present invention mayfind use in detection assays that include, but are not limited to,enzyme mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos.6,110,684, 5,958,692, 5,851,770, herein incorporated by reference intheir entireties); polymerase chain reaction; branched hybridizationmethods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246,and 5,624,802, herein incorporated by reference in their entireties);rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960and 6,235,502, herein incorporated by reference in their entireties);NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by referencein its entirety); molecular beacon technology (e.g., U.S. Pat. No.6,150,097, herein incorporated by reference in its entirety); E-sensortechnology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170,and 6,063,573, herein incorporated by reference in their entireties);cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and5,660,988, herein incorporated by reference in their entireties); DadeBehring signal amplification methods (e.g., U.S. Pat. Nos. 6,121,001,6,110,677, 5,914,230, 5,882,867, and 5,792,614, herein incorporated byreference in their entireties); ligase chain reaction (Barnay Proc.Natl. Acad. Sci. USA 88, 189-93 (1991)); and sandwich hybridizationmethods (e.g., U.S. Pat. No. 5,288,609, herein incorporated by referencein its entirety).

In some embodiments, the present invention provides kits or compositionscomprising a non-amplified oligonucleotide detection assay configuredfor detecting at least one HPV sequence. In other embodiments, thenon-amplified oligonucleotide detection assay comprises first and secondoligonucleotides configured to form an invasive cleavage structure (e.g.an INVADER assay) in combination with a target sequence comprising saidat least one HPV sequence. In particular embodiments, the firstoligonucleotide comprises a 5′ portion and a 3′ portion, wherein the 3′portion is configured to hybridize to the target sequence, and whereinthe 5′ portion is configured to not hybridize to the target sequence. Inother embodiments, the second oligonucleotide comprises a 5′ portion anda 3′ portion, wherein the 5′ portion is configured to hybridize to thetarget sequence, and wherein the 3′ portion is configured to nothybridize to the target sequence.

In some embodiments, the present invention provides a kit comprisingoligonucleotide detection assays configured for detecting a HPVsequence, wherein the kit comprises one or more oligonucleotidesselected from the group consisting of SEQ ID NOS. 1-193. In particularembodiments, the multiple HPV strains are detected simultaneously bycombining one or more of the oligonucleotides into one or morereactions. In preferred embodiments, none of the oligonucleotides arecompletely complementary to HPV target nucleic acid sequences. In someembodiments, the oligonucleotides comprise sequences not completelycomplementary to any target sequence that is detected. In preferredembodiments, all high-risk HPV strains are detected in four or fewerreactions. In other preferred embodiments, all high-risk HPV strains canbe detected in three or fewer reactions.

In some embodiments, the present invention provides a kit comprisingoligonucleotide detection assays configured for detecting all high-riskHPV strains. In preferred embodiments, the oligonucleotides are notfully complementary to nucleic acid sequences of the HPV strains. Infurther preferred embodiments, the oligonucleotides hybridize tomultiples regions of a single HPV nucleic acid (e.g., to provideredundancy in detection). In even further preferred embodiments, theoligonucleotides are selected from the group consisting of SEQ ID NOS.77-116 and 122-193.

In some embodiments, the detected HPV sequences are any of those foundbelow in Table 1 or variants thereof. It is understood that sequenceswill diverge over time and that other HPV varieties, now know, or laterdiscovered are readily adaptable to the methods and composition of thepresent invention, per the description herein.

TABLE 1 strain accession  1a NC_001356  1a U06714  2a X55964  3NC_001588  3 X74462  4 NC_001457  4 X70827  5 M17463  5 NC_001531  5bD90252  5b NC_001444  6a L41216  6a NC_001668  6b NC_001355  6 AF092932 6 NC_000904  7 M12588  7 NC_001595  7 X74463  8 M12737  8 NC_001532  9NC_001596  9 X74464 10 NC_001576 10 X74465 11 J04351 11 M14119 11NC_001525 12 NC_001577 12 X74466 13 NC_001349 13 X62843 14d NC_00157814d X74467 15 NC_001579 15 X74468 16 AF125673 16 AF472508 16 AF472509 16K02718 16 NC_001526 16 U89348 17 NC_001580 17 X74469 18 NC_001357 18X05015 18 X05349 19 NC_001581 19 X74470 20 NC_001679 20 U31778 21NC_001680 21 U31779 22 NC_001681 22 U31780 23 NC_001682 23 U31781 24NC_001683 24 U31782 25 NC_001582 25 X74471 26 NC_001583 26 X74472 27NC_001584 27 X74473 28 NC_001684 28 U31783 29 NC_001685 29 U31784 30NC_001585 30 X74474 31 J04353 31 NC_001527 32 NC_001586 32 X74475 33M12732 33 NC_001528 34 NC_001587 34 X74476 35 M74117 35 NC_001529 35hX74477 36 NC_001686 36 U31785 37 NC_001687 37 U31786 38 NC_001688 38U31787 39 M62849 39 AF548856 39 AF548857 39 NC_001535 40 NC_001589 40X74478 41 NC_001354 41 X56147 42 NC_001534 42 M73236 43 U12504 43 Y1221444 NC_001689 44 U31788 45 NC_001590 45 X74479 47 M32305 47 NC_001530 48NC_001690 48 U31789 49 NC_001591 49 X74480 50 NC_001691 50 U31790 51M62877 51 NC_001533 52 NC_001592 52 X74481 53 NC_001593 53 X74482 54AF436129 54 NC_001676 54 U37488 55 NC_001692 55 U31791 56 NC_001594 56X74483 57 NC_001353 57 X55965 57b U37537 58 D90400 58 NC_001443 59NC_001635 59 X77858 60 NC_001693 60 U31792 61 NC_001694 61 U31793 62U12499 63 NC_001458 63 X70828 64 U12495 65 NC_001459 65 X70829 66NC_001695 66 U31794 67 D21208 68 M73258 68 Y14591 69 AB027020 69NC_002171 70 NC_001711 70 U21941 71 AB040456 71 NC_002644 72 X94164 73X94165 74 AF436130 74 NC_004501 75 Y15173 76 Y15174 77 Y15175 80 Y1517682 AB027021 82 AF293961 82 NC_002172 83 AF151983 83 NC_000856 84AF293960 84 NC_002676 85 AF131950 86 AF349909 86 NC_003115 87 AJ40062887 NC_002627 89 NC_004103 90 AY057438 90 NC_004104 91 AF419318 91AF436128 91 NC_004085 92 AF531420 92 NC_004500 RXRX7 U85660

In certain embodiments, the oligonucleotide detection assays areselected from sequencing assays, polymerase chain reaction assays,hybridization assays, hybridization assays employing a probecomplementary to a mutation, microarray assays, bead array assays,primer extension assays, enzyme mismatch cleavage assays, branchedhybridization assays, rolling circle replication assays, NASBA assays,molecular beacon assays, cycling probe assays, ligase chain reactionassays, invasive cleavage structure assays, ARMS assays, and sandwichhybridization assays.

The present invention also provides methods of detecting target nucleicacids through the use of probe sequences that are not completelycomplementary to the target nucleic acid. For example, the presentinvention provides kits and methods for detecting a target sequence byusing mismatch probe sequences, comprising the steps of: a) providing asample suspected of containing a target nucleic acid; b) exposing thesample to one or more oligonucleotides that contain a region that iscomplementary to said target nucleic acid, said region having a firstportion completely complementary to said target nucleic acid, a secondportion contiguous to said first portion that is not complementary tosaid target nucleic acid (e.g., a mismatch), and a third portioncontiguous to said second portion that is completely complementary tothe target nucleic acid; and c) detecting the target nucleic acid underconditions such that no sequences that are completely complementary tothe one or more oligonucleotides or said region of the one or moreoligonucleotides are detected (i.e., only sequences that are notcompletely complementary to the oligonucleotides or the region of theoligonucleotides are detected). Thus, even if the sample containsperfect complements to the oligonucleotides or to the region, suchperfect complements are not detected. This can be carried out, forexample, through use of two or more oligonucleotides that, through theircoordinated action, provide specificity for the non-matched targetsequence, but do not detect the perfect complement. The INVADER assay,methods employing ligation, the polymerase chain reaction, etc. areexamples of methods that permit such detection. This can also be carriedout with a single probe sequence in a hybridization method if the probeis of sufficient length to ensure that it is not completelycomplementary to any sequence in the sample that might be detected.

In some embodiments, the region of the one or more oligonucleotidescontains two or more mismatches to the target nucleic acid (e.g., 3, 4,5, 6, . . . ). In some embodiments, the region contains no more thantwenty (e.g., no more than 15, 12, 10, 9, 8, 7, 6, . . . ) contiguousnucleotides that are completely complementary to the target nucleicacid. In some embodiments, one or more of the oligonucleotides aregenerated by extending a primer in an enzymatic extension reaction usingthe target nucleic acid as a template (e.g., in a polymerase chainreaction). In some embodiments, the target nucleic acid is a viraltarget nucleic acid (e.g., HPV). In some embodiments, the target nucleicacid is a conserved region of a viral genome (i.e., a region that ishighly conserved between different strains or family members of thevirus). For example, the LCR, E6, and E7 regions of the HPV genomecontain conserved sequences.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “subject” and “patient” refer to any organismsincluding plants, microorganisms and animals (e.g., mammals such asdogs, cats, livestock, and humans).

As used herein, the term “INVADER assay reagents” refers to one or morereagents for detecting target sequences, said reagents comprisingoligonucleotides capable of forming an invasive cleavage structure inthe presence of the target sequence. In some embodiments, the INVADERassay reagents further comprise an agent for detecting the presence ofan invasive cleavage structure (e.g., a cleavage agent). In someembodiments, the oligonucleotides comprise first and secondoligonucleotides, said first oligonucleotide comprising a 5′ portioncomplementary to a first region of the target nucleic acid and saidsecond oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′portion complementary to a second region of the target nucleic aciddownstream of and contiguous to the first portion. In some embodiments,the 3′ portion of the second oligonucleotide comprises a 3′ terminalnucleotide not complementary to the target nucleic acid. In preferredembodiments, the 3′ portion of the second oligonucleotide consists of asingle nucleotide not complementary to the target nucleic acid.

In some embodiments, INVADER assay reagents are configured to detect atarget nucleic acid sequence comprising first and second non-contiguoussingle-stranded regions separated by an intervening region comprising adouble-stranded region. In preferred embodiments, the INVADER assayreagents comprise a bridging oligonucleotide capable of binding to saidfirst and second non-contiguous single-stranded regions of a targetnucleic acid sequence. In particularly preferred embodiments, either orboth of said first or said second oligonucleotides of said INVADER assayreagents are bridging oligonucleotides.

In some embodiments, the INVADER assay reagents further comprise a solidsupport. For example, in some embodiments, the one or moreoligonucleotides of the assay reagents (e.g., first and/or secondoligonucleotide, whether bridging or non-bridging) is attached to saidsolid support. In some embodiments, the INVADER assay reagents furthercomprise a buffer solution. In some preferred embodiments, the buffersolution comprises a source of divalent cations (e.g., Mn²⁺ and/or Mg²⁺ions). Individual ingredients (e.g., oligonucleotides, enzymes, buffers,target nucleic acids) that collectively make up INVADER assay reagentsare termed “INVADER assay reagent components.”

In some embodiments, the INVADER assay reagents further comprise a thirdoligonucleotide complementary to a third portion of the target nucleicacid upstream of the first portion of the first target nucleic acid. Inyet other embodiments, the INVADER assay reagents further comprise atarget nucleic acid. In some embodiments, the INVADER assay reagentsfurther comprise a second target nucleic acid. In yet other embodiments,the INVADER assay reagents further comprise a third oligonucleotidecomprising a 5′ portion complementary to a first region of the secondtarget nucleic acid. In some specific embodiments, the 3′ portion of thethird oligonucleotide is covalently linked to the second target nucleicacid. In other specific embodiments, the second target nucleic acidfurther comprises a 5′ portion, wherein the 5′ portion of the secondtarget nucleic acid is the third oligonucleotide. In still otherembodiments, the INVADER assay reagents further comprise an ARRESTORmolecule (e.g., ARRESTOR oligonucleotide).

In some preferred embodiments, the INVADER assay reagents furthercomprise reagents for detecting a nucleic acid cleavage product. In someembodiments, one or more oligonucleotides in the INVADER assay reagentscomprise a label. In some preferred embodiments, said firstoligonucleotide comprises a label. In other preferred embodiments, saidthird oligonucleotide comprises a label. In particularly preferredembodiments, the reagents comprise a first and/or a thirdoligonucleotide labeled with moieties that produce a fluorescenceresonance energy transfer (FRET) effect.

In some embodiments one or more the INVADER assay reagents may beprovided in a predispensed format (i.e., premeasured for use in a stepof the procedure without re-measurement or re-dispensing). In someembodiments, selected INVADER assay reagent components are mixed andpredispensed together. In preferred embodiments, predispensed assayreagent components are predispensed and are provided in a reactionvessel (including but not limited to a reaction tube or a well, as in,e.g., a microtiter plate). In particularly preferred embodiments,predispensed INVADER assay reagent components are dried down (e.g.,desiccated or lyophilized) in a reaction vessel.

In some embodiments, the INVADER assay reagents are provided as a kit.As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to delivery systemscomprising two or more separate containers that each contains asubportion of the total kit components. The containers may be deliveredto the intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte specific reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contains a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

In some embodiments, the present invention provides INVADER assayreagent kits comprising one or more of the components necessary forpracticing the present invention. For example, the present inventionprovides kits for storing or delivering the enzymes and/or the reactioncomponents necessary to practice an INVADER assay. The kit may includeany and all components necessary or desired for assays including, butnot limited to, the reagents themselves, buffers, control reagents(e.g., tissue samples, positive and negative control targetoligonucleotides, etc.), solid supports, labels, written and/orpictorial instructions and product information, software (e.g., forcollecting and analyzing data), inhibitors, labeling and/or detectionreagents, package environmental controls (e.g., ice, desiccants, etc.),and the like. In some embodiments, the kits provide a sub-set of therequired components, wherein it is expected that the user will supplythe remaining components. In some embodiments, the kits comprise two ormore separate containers wherein each container houses a subset of thecomponents to be delivered. For example, a first container (e.g., box)may contain an enzyme (e.g., structure specific cleavage enzyme in asuitable storage buffer and container), while a second box may containoligonucleotides (e.g., INVADER oligonucleotides, probeoligonucleotides, control target oligonucleotides, etc.).

The term “label” as used herein refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) effect, andthat can be attached to a nucleic acid or protein. Labels include butare not limited to dyes; radiolabels such as ³²P; binding moieties suchas biotin; haptens such as digoxygenin; luminogenic, phosphorescent orfluorogenic moieties; mass tags; and fluorescent dyes alone or incombination with moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET). Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, characteristics of mass or behavior affected by mass (e.g.,MALDI time-of-flight mass spectrometry), and the like. A label may be acharged moiety (positive or negative charge) or alternatively, may becharge neutral. Labels can include or consist of nucleic acid or proteinsequence, so long as the sequence comprising the label is detectable.

As used herein, the term “distinct” in reference to signals refers tosignals that can be differentiated one from another, e.g., by spectralproperties such as fluorescence emission wavelength, color, absorbance,mass, size, fluorescence polarization properties, charge, etc., or bycapability of interaction with another moiety, such as with a chemicalreagent, an enzyme, an antibody, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides such asan oligonucleotide or a target nucleic acid) related by the base-pairingrules. For example, for the sequence “5′-A-G-T-3′,” is complementary tothe sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in whichonly some of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methods thatdepend upon binding between nucleic acids. Either term may also be usedin reference to individual nucleotides, especially within the context ofpolynucleotides. For example, a particular nucleotide within anoligonucleotide may be noted for its complementarity, or lack thereof,to a nucleotide within another nucleic acid strand, in contrast orcomparison to the complementarity between the rest of theoligonucleotide and the nucleic acid strand.

The term “homology” and “homologous” refers to a degree of identity.There may be partial homology or complete homology. A partiallyhomologous sequence is one that is less than 100% identical to anothersequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, and the T_(m) of the formed hybrid. “Hybridization” methodsinvolve the annealing of one nucleic acid to another, complementarynucleic acid, i.e., a nucleic acid having a complementary nucleotidesequence. The ability of two polymers of nucleic acid containingcomplementary sequences to find each other and anneal through basepairing interaction is a well-recognized phenomenon. The initialobservations of the “hybridization” process by Marmur and Lane, Proc.Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad.Sci. USA 46:461 (1960) have been followed by the refinement of thisprocess into an essential tool of modern biology.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Certain bases not commonly found innatural nucleic acids may be included in the nucleic acids of thepresent invention and include, for example, inosine and 7-deazaguanineComplementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. Several equations for calculating theT_(m) of nucleic acids are well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (see e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr.Thermodynamics and NMR of internal G. T mismatches in DNA. Biochemistry36, 10581-94 (1997) include more sophisticated computations which takestructural and environmental, as well as sequence characteristics intoaccount for the calculation of T_(m).

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of an RNA having anon-coding function (e.g., a ribosomal or transfer RNA), a polypeptideor a precursor. The RNA or polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or function is retained.

The term “wild-type” refers to a gene or a gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified”, “mutant” or “polymorphic” refers to a gene or gene productwhich displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

The term “recombinant DNA vector” as used herein refers to DNA sequencescontaining a desired heterologous sequence. For example, although theterm is not limited to the use of expressed sequences or sequences thatencode an expression product, in some embodiments, the heterologoussequence is a coding sequence and appropriate DNA sequences necessaryfor either the replication of the coding sequence in a host organism, orthe expression of the operably linked coding sequence in a particularhost organism. DNA sequences necessary for expression in prokaryotesinclude a promoter, optionally an operator sequence, a ribosome bindingsite and possibly other sequences. Eukaryotic cells are known to utilizepromoters, polyadenlyation signals and enhancers.

The term “oligonucleotide” as used herein is defined as a moleculecomprising two or more deoxyribonucleotides or ribonucleotides,preferably at least 5 nucleotides, more preferably at least about 10-15nucleotides and more preferably at least about 15 to 30 nucleotides. Theexact size will depend on many factors, which in turn depend on theultimate function or use of the oligonucleotide. The oligonucleotide maybe generated in any manner, including chemical synthesis, DNAreplication, reverse transcription, PCR, or a combination thereof. Insome embodiments, oligonucleotides that form invasive cleavagestructures are generated in a reaction (e.g., by extension of a primerin an enzymatic extension reaction).

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points towards the 5′ end of the other,the former may be called the “upstream” oligonucleotide and the latterthe “downstream” oligonucleotide. Similarly, when two overlappingoligonucleotides are hybridized to the same linear complementary nucleicacid sequence, with the first oligonucleotide positioned such that its5′ end is upstream of the 5′ end of the second oligonucleotide, and the3′ end of the first oligonucleotide is upstream of the 3′ end of thesecond oligonucleotide, the first oligonucleotide may be called the“upstream” oligonucleotide and the second oligonucleotide may be calledthe “downstream” oligonucleotide.

The term “primer” refers to an oligonucleotide that is capable of actingas a point of initiation of synthesis when placed under conditions inwhich primer extension is initiated. An oligonucleotide “primer” mayoccur naturally, as in a purified restriction digest or may be producedsynthetically.

A primer is selected to be “substantially” complementary to a strand ofspecific sequence of the template. A primer must be sufficientlycomplementary to hybridize with a template strand for primer elongationto occur. A primer sequence need not reflect the exact sequence of thetemplate. For example, a non-complementary nucleotide fragment may beattached to the 5′ end of the primer, with the remainder of the primersequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

The term “cleavage structure” as used herein, refers to a structure thatis formed by the interaction of at least one probe oligonucleotide and atarget nucleic acid, forming a structure comprising a duplex, theresulting structure being cleavable by a cleavage means, including butnot limited to an enzyme. The cleavage structure is a substrate forspecific cleavage by the cleavage means in contrast to a nucleic acidmolecule that is a substrate for non-specific cleavage by agents such asphosphodiesterases which cleave nucleic acid molecules without regard tosecondary structure (i.e., no formation of a duplexed structure isrequired).

The term “cleavage means” or “cleavage agent” as used herein refers toany means that is capable of cleaving a cleavage structure, includingbut not limited to enzymes. “Structure-specific nucleases” or“structure-specific enzymes” are enzymes that recognize specificsecondary structures in a nucleic molecule and cleave these structures.The cleavage means of the invention cleave a nucleic acid molecule inresponse to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

The cleavage means may include nuclease activity provided from a varietyof sources including the Cleavase enzymes, the FEN-1 endonucleases(including RAD2 and XPG proteins), Taq DNA polymerase and E. coli DNApolymerase I. The cleavage means may include enzymes having 5′ nucleaseactivity (e.g., Taq DNA polymerase (DNAP), E. coli DNA polymerase I).The cleavage means may also include modified DNA polymerases having 5′nuclease activity but lacking synthetic activity. Examples of cleavagemeans suitable for use in the method and kits of the present inventionare provided in U.S. Pat. Nos. 5,614,402; 5,795,763; 5,843,669; 6,090;PCT Appln. Nos WO 98/23774; WO 02/070755A2; and WO0190337A2, each ofwhich is herein incorporated by reference it its entirety.

The term “thermostable” when used in reference to an enzyme, such as a5′ nuclease, indicates that the enzyme is functional or active (i.e.,can perform catalysis) at an elevated temperature, i.e., at about 55° C.or higher.

The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i.e., the treatment of a cleavage structure with a cleavage means).

The term “target nucleic acid” refers to a nucleic acid moleculecontaining a sequence that has at least partial complementarity with atleast a probe oligonucleotide and may also have at least partialcomplementarity with an INVADER oligonucleotide. The target nucleic acidmay comprise single- or double-stranded DNA or RNA.

The term “non-target cleavage product” refers to a product of a cleavagereaction that is not derived from the target nucleic acid. As discussedabove, in the methods of the present invention, cleavage of the cleavagestructure generally occurs within the probe oligonucleotide. Thefragments of the probe oligonucleotide generated by this target nucleicacid-dependent cleavage are “non-target cleavage products.”

The term “probe oligonucleotide” refers to an oligonucleotide thatinteracts with a target nucleic acid to form a cleavage structure in thepresence or absence of an INVADER oligonucleotide. When annealed to thetarget nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.

The term “INVADER oligonucleotide” refers to an oligonucleotide thathybridizes to a target nucleic acid at a location near the region ofhybridization between a probe and the target nucleic acid, wherein theINVADER oligonucleotide comprises a portion (e.g., a chemical moiety, ornucleotide—whether complementary to that target or not) that overlapswith the region of hybridization between the probe and target. In someembodiments, the INVADER oligonucleotide contains sequences at its 3′end that are substantially the same as sequences located at the 5′ endof a probe oligonucleotide.

The term “cassette” as used herein refers to an oligonucleotide orcombination of oligonucleotides configured to generate a detectablesignal in response to cleavage of a probe oligonucleotide in an INVADERassay. In preferred embodiments, the cassette hybridizes to a non-targetcleavage product from cleavage of the probe oligonucleotide to form asecond invasive cleavage structure, such that the cassette can then becleaved.

In some embodiments, the cassette is a single oligonucleotide comprisinga hairpin portion (i.e., a region wherein one portion of the cassetteoligonucleotide hybridizes to a second portion of the sameoligonucleotide under reaction conditions, to form a duplex). In otherembodiments, a cassette comprises at least two oligonucleotidescomprising complementary portions that can form a duplex under reactionconditions. In preferred embodiments, the cassette comprises a label. Inparticularly preferred embodiments, cassette comprises labeled moietiesthat produce a fluorescence resonance energy transfer (FRET) effect.

The term “substantially single-stranded” when used in reference to anucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

As used herein, the phrase “non-amplified oligonucleotide detectionassay” refers to a detection assay configured to detect the presence orabsence of a particular polymorphism (e.g., SNP, repeat sequence, etc.)in a target sequence (e.g. genomic DNA) that has not been amplified(e.g. by PCR), without creating copies of the target sequence. A“non-amplified oligonucleotide detection assay” may, for example,amplify a signal used to indicate the presence or absence of aparticular polymorphism in a target sequence, so long as the targetsequence is not copied.

The term “sequence variation” as used herein refers to differences innucleic acid sequence between two nucleic acids. For example, awild-type structural gene and a mutant form of this wild-type structuralgene may vary in sequence by the presence of single base substitutionsand/or deletions or insertions of one or more nucleotides. These twoforms of the structural gene are said to vary in sequence from oneanother. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

The term “liberating” as used herein refers to the release of a nucleicacid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of, for example, a 5′ nuclease such thatthe released fragment is no longer covalently attached to the remainderof the oligonucleotide.

The term “K_(m)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides including but not limited to analogsthat have altered stacking interactions such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP); base analogs with alternative hydrogenbonding configurations (e.g., such as Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983 to S.Benner); non-hydrogen bonding analogs (e.g., non-polar, aromaticnucleoside analogs such as 2,4-difluorotoluene, described by B. A.Schweitzer and E. T. Kool, J. Org. Chem., 1994, 59, 7238-7242, B. A.Schweitzer and E. T. Kool, J. Am. Chem. Soc., 1995, 117, 1863-1872);“universal” bases such as 5-nitroindole and 3-nitropyrrole; anduniversal purines and pyrimidines (such as “K” and “P” nucleotides,respectively; P. Kong, et al., Nucleic Acids Res., 1989, 17,10373-10383, P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152).Nucleotide analogs include comprise modified forms ofdeoxyribonucleotides as well as ribonucleotides.

The term “polymorphic locus” is a locus present in a population thatshows variation between members of the population (e.g., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

The term “microorganism” as used herein means an organism too small tobe observed with the unaided eye and includes, but is not limited tobacteria, virus, protozoans, fungi, and ciliates.

The term “microbial gene sequences” refers to gene sequences derivedfrom a microorganism.

The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

As used herein, the terms “high-risk HPV strains” or “high-risk HPVtypes” refer to those strains of HPV that have been found in cancers(e.g., carcinomas). These HPV strains include HPV types 16, 18, 30, 31,33, 35, 39, 45, 51, 52, 56, 58, 59, 67, 68, 69 and 70.

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

The term “multi-drug resistant” or multiple-drug resistant” refers to amicroorganism that is resistant to more than one of the antibiotics orantimicrobial agents used in the treatment of said microorganism.

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples. A sample mayinclude a specimen of synthetic origin.

Biological samples may be animal, including human, fluid, solid (e.g.,stool) or tissue, as well as liquid and solid food and feed products andingredients such as dairy items, vegetables, meat and meat by-products,and waste. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,lagamorphs, rodents, etc.

Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable and non-disposable items. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

The term “source of target nucleic acid” refers to any sample thatcontains nucleic acids (RNA or DNA). Particularly preferred sources oftarget nucleic acids are biological samples including, but not limitedto blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph,sputum and semen.

An oligonucleotide is said to be present in “excess” relative to anotheroligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

A sample “suspected of containing” a first and a second target nucleicacid may contain either, both or neither target nucleic acid molecule.

The term “reactant” is used herein in its broadest sense. The reactantcan comprise, for example, an enzymatic reactant, a chemical reactant orlight (e.g., ultraviolet light, particularly short wavelengthultraviolet light is known to break oligonucleotide chains). Any agentcapable of reacting with an oligonucleotide to either shorten (i.e.,cleave) or elongate the oligonucleotide is encompassed within the term“reactant.”

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, recombinant CLEAVASEnucleases are expressed in bacterial host cells and the nucleases arepurified by the removal of host cell proteins; the percent of theserecombinant nucleases is thereby increased in the sample.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid (e.g., 4, 5, 6, . . . , n−1).

The term “nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin that may besingle or double stranded, and represent the sense or antisense strand.Similarly, “amino acid sequence” as used herein refers to peptide orprotein sequence.

As used herein, the terms “purified” or “substantially purified” referto molecules, either nucleic or amino acid sequences, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” or “isolated oligonucleotide” is therefore asubstantially purified polynucleotide.

The term “continuous strand of nucleic acid” as used herein is means astrand of nucleic acid that has a continuous, covalently linked,backbone structure, without nicks or other disruptions. The dispositionof the base portion of each nucleotide, whether base-paired,single-stranded or mismatched, is not an element in the definition of acontinuous strand. The backbone of the continuous strand is not limitedto the ribose-phosphate or deoxyribose-phosphate compositions that arefound in naturally occurring, unmodified nucleic acids. A nucleic acidof the present invention may comprise modifications in the structure ofthe backbone, including but not limited to phosphorothioate residues,phosphonate residues, 2′ substituted ribose residues (e.g., 2′-O-methylribose) and alternative sugar (e.g., arabinose) containing residues.

The term “continuous duplex” as used herein refers to a region of doublestranded nucleic acid in which there is no disruption in the progressionof basepairs within the duplex (i.e., the base pairs along the duplexare not distorted to accommodate a gap, bulge or mismatch with theconfines of the region of continuous duplex). As used herein the termrefers only to the arrangement of the basepairs within the duplex,without implication of continuity in the backbone portion of the nucleicacid strand. Duplex nucleic acids with uninterrupted basepairing, butwith nicks in one or both strands are within the definition of acontinuous duplex.

The term “duplex” refers to the state of nucleic acids in which the baseportions of the nucleotides on one strand are bound through hydrogenbonding the their complementary bases arrayed on a second strand. Thecondition of being in a duplex form reflects on the state of the basesof a nucleic acid. By virtue of base pairing, the strands of nucleicacid also generally assume the tertiary structure of a double helix,having a major and a minor groove. The assumption of the helical form isimplicit in the act of becoming duplexed.

The term “template” refers to a strand of nucleic acid on which acomplementary copy is built from nucleoside triphosphates through theactivity of a template-dependent nucleic acid polymerase. Within aduplex the template strand is, by convention, depicted and described asthe “bottom” strand. Similarly, the non-template strand is oftendepicted and described as the “top” strand.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of INVADER oligonucleotides, probeoligonucleotides and FRET cassettes for detecting a two differentalleles (e.g., differing by a single nucleotide) in a single reaction.

FIG. 2 shows the results of a temperature optimization experimentcarried out in some embodiments of the present invention.

FIGS. 3A and 3B show sequences of detection assay components in someembodiments of the present invention. Underlined portions of thesequence refer to the 5′ arm portion of probe oligonucleotides.

FIG. 4 shows results of HPV strain 16 detection experiments conducted insome embodiments of the present invention.

FIG. 5 shows results of HPV strain 18 detection experiments conducted insome embodiments of the present invention.

FIG. 6 shows HPV strains detected with Invader assay pools A9, A7 andA5/A6.

FIGS. 7A and 7B show sequences of detection assay components in someembodiments of the present invention. Underlined portions of thesequence refer to the 5′ arm portion of probe oligonucleotides.

FIG. 8 shows the quantitation of cervical sample genomic DNA using theOligreen Quantitation Kit or the Alpha-Actin Invader assays.

FIG. 9 shows detection of HPV and Alpha-Actin in cervical samplesconducted in some embodiments of the present invention.

FIGS. 10A-C show sequences of detection assay components in someembodiments of the present invention. Underlined portions of thesequence refer to the 5′ arm portion of probe oligonucleotides.

DESCRIPTION OF THE INVENTION

The present invention provides means for forming a nucleic acid cleavagestructure that is dependent upon the presence of a target nucleic acidand cleaving the nucleic acid cleavage structure so as to releasedistinctive cleavage products. 5′ nuclease activity, for example, isused to cleave the target-dependent cleavage structure and the resultingcleavage products are indicative of the presence of specific targetnucleic acid sequences in the sample. When two strands of nucleic acid,or oligonucleotides, both hybridize to a target nucleic acid strand suchthat they form an overlapping invasive cleavage structure, as describedbelow, invasive cleavage can occur. Through the interaction of acleavage agent (e.g., a 5′ nuclease) and the upstream oligonucleotide,the cleavage agent can be made to cleave the downstream oligonucleotideat an internal site in such a way that a distinctive fragment isproduced. Such embodiments have been termed the INVADER assay (ThirdWave Technologies) and are described in U.S. Pat. Nos. 5,846,717,5,985,557, 5,994,069, 6,001,567, and 6,090,543, WO 97/27214 WO 98/42873,Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA,97:8272 (2000), each of which is herein incorporated by reference intheir entirety for all purposes).

The INVADER assay detects hybridization of probes to a target byenzymatic cleavage of specific structures by structure specific enzymes(See, INVADER assays, Third Wave Technologies; See e.g., U.S. Pat. Nos.5,846,717; 6,090,543; 6,001,567; 5,985,557; 6,090,543; 5,994,069;Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA,97:8272 (2000), WO97/27214 and WO98/42873, each of which is hereinincorporated by reference in their entirety for all purposes).

The INVADER assay detects specific DNA and RNA sequences by usingstructure-specific enzymes (e.g. FEN endonucleases) to cleave a complexformed by the hybridization of overlapping oligonucleotide probes (See,e.g. FIG. 1). Elevated temperature and an excess of one of the probesenable multiple probes to be cleaved for each target sequence presentwithout temperature cycling. In some embodiments, these cleaved probesthen direct cleavage of a second labeled probe. The secondary probeoligonucleotide can be 5′-end labeled with fluorescein that is quenchedby an internal dye. Upon cleavage, the de-quenched fluorescein labeledproduct may be detected using a standard fluorescence plate reader.

The INVADER assay detects specific mutations and SNPs in unamplified, aswell as amplified, RNA and DNA including genomic DNA. In the embodimentsshown schematically in FIG. 1, the INVADER assay uses two cascadingsteps (a primary and a secondary reaction) both to generate and then toamplify the target-specific signal. For convenience, the alleles in thefollowing discussion are described as wild-type (WT) and mutant (MT),even though this terminology does not apply to all genetic variations.In the primary reaction (FIG. 1, panel A), the WT primary probe and theINVADER oligonucleotide hybridize in tandem to the target nucleic acidto form an overlapping structure. An unpaired “flap” is included on the5′ end of the WT primary probe. A structure-specific enzyme (e.g. theCLEAVASE enzyme, Third Wave Technologies) recognizes the overlap andcleaves off the unpaired flap, releasing it as a target-specificproduct. In the secondary reaction, this cleaved product serves as anINVADER oligonucleotide on the WT fluorescence resonance energy transfer(WT-FRET) probe to again create the structure recognized by thestructure specific enzyme (panel A). When the two dyes on a single FRETprobe are separated by cleavage (indicated by the arrow in FIG. 1), adetectable fluorescent signal above background fluorescence is produced.Consequently, cleavage of this second structure results in an increasein fluorescence, indicating the presence of the WT allele (or mutantallele if the assay is configured for the mutant allele to generate thedetectable signal). In some embodiments, FRET probes having differentlabels (e.g. resolvable by difference in emission or excitationwavelengths, or resolvable by time-resolved fluorescence detection) areprovided for each allele or locus to be detected, such that thedifferent alleles or loci can be detected in a single reaction. In suchembodiments, the primary probe sets and the different FRET probes may becombined in a single assay, allowing comparison of the signals from eachallele or locus in the same sample.

If the primary probe oligonucleotide and the target nucleotide sequencedo not match perfectly at the cleavage site (e.g., as with the MTprimary probe and the WT target, FIG. 1, panel B), the overlappedstructure does not form and cleavage is suppressed. The structurespecific enzyme (e.g., CLEAVASE VIII enzyme, Third Wave Technologies)used cleaves the overlapped structure more efficiently (e.g. at least340-fold) than the non-overlapping structure, allowing excellentdiscrimination of the alleles.

The probes turn over without temperature cycling to produce many signalsper target (i.e., linear signal amplification). Similarly, eachtarget-specific product can enable the cleavage of many FRET probes.

The primary INVADER assay reaction is directed against the target DNA(or RNA) being detected. The target DNA is the limiting component in thefirst invasive cleavage, since the INVADER and primary probe aresupplied in molar excess. In the second invasive cleavage, it is thereleased flap that is limiting. When these two cleavage reactions areperformed sequentially, the fluorescence signal from the compositereaction accumulates linearly with respect to the target DNA amount.

In certain embodiments, the INVADER assay, or other nucleotide detectionassays, are performed with accessible site designed oligonucleotidesand/or bridging oligonucleotides. Such methods, procedures andcompositions are described in U.S. Pat. No. 6,194,149, WO9850403, andWO0198537, all of which are specifically incorporated by reference intheir entireties.

In certain embodiments, the target nucleic acid sequence is amplifiedprior to detection (e.g. such that synthetic nucleic acid is generated).In some embodiments, the target nucleic acid comprises genomic DNA. Inother embodiments, the target nucleic acid comprises synthetic DNA orRNA. In some preferred embodiments, synthetic DNA within a sample iscreated using a purified polymerase. In some preferred embodiments,creation of synthetic DNA using a purified polymerase comprises the useof PCR. In other preferred embodiments, creation of synthetic DNA usinga purified DNA polymerase, suitable for use with the methods of thepresent invention, comprises use of rolling circle amplification, (e.g.,as in U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502, hereinincorporated by reference in their entireties). In other preferredembodiments, creation of synthetic DNA comprises copying genomic DNA bypriming from a plurality of sites on a genomic DNA sample. In someembodiments, priming from a plurality of sites on a genomic DNA samplecomprises using short (e.g., fewer than about 8 nucleotides)oligonucleotide primers. In other embodiments, priming from a pluralityof sites on a genomic DNA comprises extension of 3′ ends in nicked,double-stranded genomic DNA (i.e., where a 3′ hydroxyl group has beenmade available for extension by breakage or cleavage of one strand of adouble stranded region of DNA). Some examples of making synthetic DNAusing a purified polymerase on nicked genomic DNAs, suitable for usewith the methods and compositions of the present invention, are providedin U.S. Pat. No. 6,117,634, issued Sep. 12, 2000, and U.S. Pat. No.6,197,557, issued Mar. 6, 2001, and in PCT application WO 98/39485, eachincorporated by reference herein in their entireties for all purposes.

In some embodiments, the present invention provides methods fordetecting a target sequence, comprising: providing a) a samplecontaining DNA amplified by extension of 3′ ends in nickeddouble-stranded genomic DNA, said genomic DNA suspected of containingsaid target sequence; b) oligonucleotides capable of forming an invasivecleavage structure in the presence of said target sequence; and c)exposing the sample to the oligonucleotides and the agent. In someembodiments, the agent comprises a cleavage agent. In some particularlypreferred embodiments, the method of the invention further comprises thestep of detecting said cleavage product.

In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

In some particularly preferred embodiments, the target sequencecomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, and saidoligonucleotides comprise first and second oligonucleotides, saidwherein at least a portion of said first oligonucleotide is completelycomplementary to said first portion of said target sequence and whereinsaid second oligonucleotide comprises a 3′ portion and a 5′ portion,wherein said 5′ portion is completely complementary to said secondportion of said target nucleic acid.

In other embodiments, synthetic DNA suitable for use with the methodsand compositions of the present invention is made using a purifiedpolymerase on multiply-primed genomic DNA, as provided, e.g., in U.S.Pat. Nos. 6,291,187, and 6,323,009, and in PCT applications WO 01/88190and WO 02/00934, each herein incorporated by reference in theirentireties for all purposes. In these embodiments, amplification of DNAsuch as genomic DNA is accomplished using a DNA polymerase, such as thehighly processive Φ 29 polymerase (as described, e.g., in U.S. Pat. Nos.5,198,543 and 5,001,050, each herein incorporated by reference in theirentireties for all purposes) in combination with exonuclease-resistantrandom primers, such as hexamers.

In some embodiments, the present invention provides methods fordetecting a target sequence, comprising: providing a) a samplecontaining DNA amplified by extension of multiple primers on genomicDNA, said genomic DNA suspected of containing said target sequence; b)oligonucleotides capable of forming an invasive cleavage structure inthe presence of said target sequence; and c) exposing the sample to theoligonucleotides and the agent. In some embodiments, the agent comprisesa cleavage agent. In some preferred embodiments, said primers are randomprimers. In particularly preferred embodiments, said primers areexonuclease resistant. In some particularly preferred embodiments, themethod of the invention further comprises the step of detecting saidcleavage product.

In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

In some preferred embodiments, the exposing of the sample to theoligonucleotides and the agent comprises exposing the sample to theoligonucleotides and the agent under conditions wherein an invasivecleavage structure is formed between said target sequence and saidoligonucleotides if said target sequence is present in said sample,wherein said invasive cleavage structure is cleaved by said cleavageagent to form a cleavage product.

In some particularly preferred embodiments, the target sequencecomprises a first region and a second region, said second regiondownstream of and contiguous to said first region, and saidoligonucleotides comprise first and second oligonucleotides, saidwherein at least a portion of said first oligonucleotide is completelycomplementary to said first portion of said target sequence and whereinsaid second oligonucleotide comprises a 3′ portion and a 5′ portion,wherein said 5′ portion is completely complementary to said secondportion of said target nucleic acid.

In certain embodiments, the present invention provides kits for assayinga pooled sample (e.g., a pooled blood sample) using INVADER detectionreagents (e.g. primary probe, INVADER probe, and FRET cassette). Inpreferred embodiments, the kit further comprises instructions on how toperform the INVADER assay and specifically how to apply the INVADERdetection assay to pooled samples from many individuals, or to “pooled”samples from many cells (e.g. from a biopsy sample) from a singlesubject.

The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage of the probeswithout the need to use temperature cycling (i.e., for periodicdenaturation of target nucleic acid strands) or nucleic acid synthesis(i.e., for the polymerization-based displacement of target or probenucleic acid strands). When a cleavage reaction is run under conditionsin which the probes are continuously replaced on the target strand (e.g.through probe-probe displacement or through an equilibrium betweenprobe/target association and disassociation, or through a combinationcomprising these mechanisms, (The kinetics of oligonucleotidereplacement. Luis P. Reynaldo, Alexander V. Vologodskii, Bruce P. Neriand Victor I. Lyamichev. J. Mol. Biol. 97: 511-520 (2000)), multipleprobes can hybridize to the same target, allowing multiple cleavages,and the generation of multiple cleavage products.

In some embodiments, the detection assays of the present invention aredesigned to detect one or more HPV sequences (See, e.g., Example 5). Insome embodiments, multiple HPV sequences are detected in a singlereaction (See, e.g., Example 5, FIG. 9, reactions 10-658, 10-662, 10-677and 10-682). In some preferred embodiments, a single oligonucleotideused in the detection assays is configured to hybridize to two or moreHPV sequences such that multiple HPV sequences can be detected with asingle set of detection assay reagents (See, e.g., Example 5, FIG. 9).In some embodiments, the oligonucleotides used in the detection assayare perfectly complementary to the intended HPV target sequence. Inother embodiments, the oligonucleotides contain one or more mismatchesto the HPV target sequence of interest. Mismatches find multiple uses,including, but not limited to, the ability to reduce hybridizationefficiency (which may be desired in some detection assay formats), theability to add degeneracy (e.g., to detect two or more strains orvariants), and the ability to compensate for sequence variation that maybe in a sample. In some embodiments, where variation at a particularnucleotide position is identified in some members of a testedpopulation, multiple oligonucleotides are provides that differ insequence at the position so that each variant within the population isdetected. Exemplary detection assay components for use in invasivecleavage assays are provided in the Example section below for certainpreferred strains of HPV.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); DS (dextran sulfate); ° C. (degrees Centigrade); and Sigma(Sigma Chemical Co., St. Louis, Mo.).

Example 1 Design of Oligonucleotides to Detect Multiple HPV Strains

The objective of these experiments was to arrive at oligonucleotidedesigns suitable for use in INVADER assays comprising multiple targetHPV strains. As a first step, HPV DNA sequences were obtained fromGenbank and aligned using SEQ WEB GAP and PRETTY programs (Accelrys, SanDiego, Calif.). Only those regions of HPV that are reported to remainintact following chromosomal integration were analyzed to permit theassays to detect both integrated and non-integrated HPV sequences.Regions of suitable sequence conservation were chosen for select groupsof strains. In this example, areas within the LCR, E6, and E7 genes werefound to have considerable homology between HPV 18 and 59.

Candidate probe oligonucleotides were designed by searching forstretches of sequence comprising a limited number of mismatches betweenthe two targets in either pair. Designs were generated to severalsequences on either the sense or antisense strands. Suitable INVADERoligonucleotides were designed to accompany the respective probeoligonucleotide candidates. Initial INVADER oligonucleotide designs wereselected to associate with only a single target, e.g. HPV 18, HPV 45, orHPV 59; subsequent designs hybridize to more than one HPV strain.Candidate probe sets were then evaluated for two types of performancecriteria: (1) signal generation at the chosen reaction temperature and(2) limit of detection, i.e. signal over background ratios at low levelsof target DNA. Probe sets meeting desired performance cut-offs, in thiscase, optimal signal generation at 63° C. and LOD of ≦1000 copies of HPVDNA, were then selected for further evaluation.

Temperature Optimization

INVADER assays were performed in 96 well MJ Skirted microtiter plates.Plates were incubated using either an MJ Research PTC100 Thermocycler ora ThermoHybaid PCR Express (Molecular Biology Instrumentation, NeedhamHeights, Mass.) and read with an Applied Biosystems CYTOFLUOR® 4000series multiwell plate reader.

INVADER assays to determine temperature optima of probe sets were set upby preparing primary and secondary reaction master mixes. In theseexperiments, two different INVADER oligonucleotides were tested incombination with a single probe oligonucleotide in each reaction. Forexample, in experiments designed to test probe sets for HPV 18 and 59,INVADER oligonucleotides for both HPV 18 and HPV 59 were included ineach reaction along with a single probe oligonucleotide designed toassociate with both strains of HPV. These reaction mixtures were testedseparately on plasmid DNA comprising a portion of the HPV 18 (18c1)sequence (ATCC Catalog Number: 45152D) and a synthetic target comprisinga portion of the HPV 59 sequence (SEQ ID NO: 42). Similarly, inexperiments designed to detect HPV 45 and 59, INVADER oligonucleotidesfor both strains were included in each reaction along with thecorresponding probe oligonucleotide and appropriate controls.

Master mixes containing primary reaction components were assembled foreach set of temperature optimization reactions as follows. Reactionswere carried out in parallel in microtiter plates.

Primary Mix (PM) Stock Final # Total Reagent Conc. Conc. Rxns Vol/RxnVol Invader Oligo 10 0.05 120 0.10 12 1 (μM) Invader Oligo 10 0.05 0.1012 2 (μM) Primary 10 0.5 1.00 120 Probe (μM) MOPS (mM) 400 10 0.50 60CLEAVASE 50 2.5 1.00 120 X enzyme (ng/μl) MgCl₂ (mM) 250 12.5 1.00 120Distilled 0 0 0.30 36 Water 4.00 μl/rxn

Aliquots of 15 μl of each target at a concentration of 20 fM were placedin the appropriate wells of a microtiter plate and were overlaid with 20μl of mineral oil; 20 μl of 10 ng/μl tRNA were used for the no targetcontrol reactions. All reactions were run in duplicate. The targets wereheat denatured at 95° C. for 5 minutes, cooled to 20° C., and thenaliquots of 4 μl of the primary mix were added to each well. Themicrotiter plates were incubated for 2 hours in a ThermoHybaidthermocycler with a gradient heat block over a span of 10 degrees (i.e.reactions were run at 58, 58.3, 58.9, 59.8, 60.9, 62.2, 63.5, 64.9,66.4, 67.3, 67.8, 68.1° C.) and then returned to 20° C.

A secondary master mix (SM) was assembled as follows.

Secondary Mix (SM) Stock Final Reagent Conc. Conc. Vol/Rxn Total VolFRET Cassette 10 0.25 0.50 62.50 Arm 1 FAM SEQ ID NO: 63 (μM) MOPS (mM)400 0.91 0.05 5.68 Water — — 2.45 306.82 3 μl/rxn

Aliquots of 3 μl of secondary mix were then added to each well, and theplate was incubated at 63° C. for 10 minutes and then cooled to 4° C.prior to scanning in a CYTOFLUOR 4000 fluorescence plate reader (AppliedBiosystems, Foster City, Calif.). The settings used were: 485/20 nmexcitation/bandwidth and 530/25 nm emission/bandwidth for FAM dyedetection. Unlike typical biplex INVADER reactions, because these assaysinclude only a single probe molecule, only the single corresponding FRETcassette is required. The instrument gain was set for each dye so thatthe No Target Blank produced between 50-150 Relative Fluorescence Units(RFUs).

Because the optimal gain setting can vary between instruments, gain isadjusted as needed to give the best signal/background ratio (sample rawsignal divided by the No Target Control signal) or No Target Controlsample readings of ˜100 RFUs. Fluorescence microplate readers that use axenon lamp source generally produce higher RFUs. For directly readingthe microplates, the probe height of, and how the plate is positionedin, the fluorescence microplate reader may need to be adjusted accordingto the manufacturer's recommendations.

The raw data that is generated by the device/instrument is used tomeasure the assay performance (real-time or endpoint mode). Theequations below provide how FOZ (Fold Over Zero), and other values arecalculated. NTC in the equations below represents the signal from the NoTarget Control.

FOZ or Signal/No TargetFOZ_(Dye1)=(RawSignal_(Dye1)/NTC_(Dye1))

Candidate probe sets were selected based on the temperature profilesgenerated in these experiments. In particular, desirable probe setsexhibit temperature profiles on the two targets (e.g. HPV 18 and 59)tested together that exhibit similar trends with respect to increase intemperature, typically a bell shaped curve with its peak at the chosenreaction temperature, in this case 63° C. An additional desirablefeature is that the peak not be precipitously lower plus or minus 1 or 2degrees from 63° C. Fewer than 30% of the candidate probe sets yieldedsuitable temperature profiles.

In order to unify reaction conditions at a single reaction temperature,probe designs that gave rise to similar trends in response totemperature were chosen for further design optimization. Redesignedprobes in which probe length was altered were tested. FIG. 2 shows theresults of a temperature optimization experiment carried out with probeT3e1b (SEQ ID NO:39) and INVADER oligonucleotides designed to detect HPV18 (T3e4i) (SEQ ID NO:40) and HPV 59 (T3e6i) (SEQ ID NO:41),respectively, on both the HPV 18 plasmid and HPV 59 (T3rT59) synthetictarget (SEQ ID NO:42).

Similar temperature optimization and redesign procedures were carriedout for all of the oligonucleotides presented in FIGS. 3 and 7.

Limit of Detection (LOD) Analysis

In addition to optimizing for temperature profiles that follow the samegeneral trends in response to temperature and do not present steepslopes in the immediate vicinity of the target reaction temperature, itis also desirable to optimize probe sets for analytical sensitivity orlimit of detection (LOD). Measuring LOD is accomplished by conductingINVADER assays at a single reaction temperature while varying targetconcentration.

Reactions to determine LOD of temperature optimized probe sets were setup as follows. A dilution series of target DNAs (HPV 18 plasmid andsynthetic target SEQ ID NO: 42) was made in 10 ng/μl tRNA in dH₂O; inthe example presented here, target amounts per assay ranged from 125copies/rxn to 8000 copies/rxn, doubling in each successive reaction.Aliquots of 15 μl of diluted target or 10 ng/μl tRNA in dH₂O for the notarget controls were placed in appropriate wells of a microtiter plateand overlaid with 20 μl of mineral oil. All reactions were run inquadruplicate. A master mix (MM) was made containing buffer, CLEAVASE Xenzyme, MgCl₂, both INVADER oligonucleotides (SEQ ID NOs: 40-41),primary probe T3e1b (SEQ ID NO: 39) and FRET cassette oligonucleotides(SEQ ID NO: 63) as below.

Master Mix (MM): no. 125 rxns Stock Final Vol/ Total Reagent Conc. Conc.Rxn Vol FRET Cassette (μM) 10 0.25 0.5 62.5 MOPS (mM) 400 10 0.5 62.5CLEAVASE X enzyme 50 2.5 1 125 (ng/μl) MgCl2 (mM) 250 12.5 1 125 Invaderoligo 1 10 0.05 0.1 12.5 (μM) Invader oligo 2 10 0.05 0.1 12.5 (uM)Primary Probe 10 0.5 1 125 (μM) water N/A N/A 0.8 100 total volume 5 625

Microtiter plates were covered and incubated at 95° C. for 5 minutes todenature the targets and then cooled to 20° C. Aliquots of 5 μl ofmaster mix were added and the reactions heated to 63° C. for 4 hours.Upon completion, plates were removed to the CYTOFLUOR plate reader andanalyzed as described above. Representative results are presented FIG.2. These results demonstrate that the designs tested in this experimentare suitable for the detection of as few as 250 copies of thecorresponding HPV 18 and 59 sequences.

Example 2 Design of Oligonucleotides to Detect HPV 16

Candidate oligonucleotide sets having a primary probe and an INVADERoligonucleotide were designed to detect regions in both HPV 16 and HPV31 using the procedures described in the preceding examples. Designswere directed to the E7 gene of HPV. As in Example 1, different INVADERoligonucleotide sequences were tested in combination with a single probesequence to find a probe set with optimal performance characteristics atthe desired reaction temperature (63° C.) and in terms of limit ofdetection (FOZ). Both temperature optimization experiments and LOD (FOZ)experiments were conducted as described above using 15 μl of a 20 fMstock solution of HPV 16 plasmid (ATCC Catalog Number: 45113D). A totalof 24 different INVADER oligonucleotides were tested with SEQ ID NO: 1(Alg3p); of these, one was chosen for use in assays to detect HPV 16:SEQ ID NO: 2 (Alg3p), based on its temperature optimization profile andFOZ.

Co-Detection of HPV 16 and a Human Genomic Internal Control Sequence

In some applications, it is desirable to co-detect an internal controlsequence, for example in order to determine whether or not there aresample inhibition effects or operator errors. Oligonucleotide sets weredesigned to detect three different human genomic sequences and weretested in three different biplexed INVADER assays in combination withSEQ ID NOs: 1 and 2 to detect the HPV 16 plasmid. The human genomicregions were alpha actin (Genbank accession number NM_(—)001100), the 3′untranslated region (UTR) of CFTR (Genbank accession numberNM_(—)000492), and hIGF (Genbank accession number AY260957). Theoligonucleotides used for these designs were developed previously andoptimized as described in the previous examples and were as follows:alpha actin probe SEQ ID NO: 64, INVADER oligo, SEQ ID NO: 65, FRETcassette 68; 3′ UTR CFTR probe SEQ ID NO: 66, INVADER oligo SEQ ID NO:67, FRET cassette SEQ ID NO: 68; hIGF probe SEQ ID NO: 69, INVADER oligoSEQ ID NO: 70, FRET cassette SEQ ID NO: 71.

A standard curve was generated using different amounts of HPV 16 plasmidagainst a constant amount of human genomic DNA. Reactions containing 0,250, 500, 1000, 2500, 5000, 10,000, or 20,000 copies of the HPV 16plasmid and either 100 ng (for hIGF and CFTR) or 250 ng (for alphaactin) human genomic DNA. DNA was isolated using the Gentra PUREGENE®Autopure LS system (Gentra, Inc., Minneapolis, Minn.) or manualpreparation methods. All other reaction components and detection were asdescribed in the previous examples except that a second FRET oligo wasused in each case (for hIGF, SEQ ID NO: 71, red dye; for 3′ UTR of CFTRand alpha actin, SEQ ID NO: 68, red dye). The results are presented inFIG. 4 and indicate that all of the human genomic sequences tested weresuitable for biplex detection in combination with varying levels of HPV16 plasmid DNA. Furthermore, these experiments demonstrate that there isno apparent cross reactivity between the probe sets designed to detectHPV 16 and those designed to detect the human genomic sequences, asevidenced by both the unchanged signal generated using the IC probes inthe presence of variable amounts of HPV 16 DNA as well as by the lack ofdetectable signal generated using the HPV 16 probes in the absence ofHPV 16 plasmid DNA.

Example 3 Effects of Genomic DNA on Detection of HPV 18

Experiments were carried out to assess the effect of exogenous humangenomic DNA on detection of HPV 18. INVADER reactions were set up asfollows. Serial dilutions of a synthetic HPV 18 target B1T18 (SEQ ID NO:72) were made to result in numbers of target molecules as indicated inthe X-axis of FIG. 5 when 15 μl were pipetted into the appropriate wellsof a microtiter plate. A second set of serial dilutions was madeincorporating human genomic DNA, purified as described in Example 2,into each dilution such that each reaction contained 1 μg of humangenomic DNA. No target controls contained 15 μl of 10 ng/μl tRNA indistilled water. All reactions were run in duplicate. The targetaliquots were overlaid with 20 μl mineral oil and denatured at 95° C.for 5 minutes then chilled to 20° C. A master mix containing INVADERreaction components was made as follows.

Stock Final # of Volume/ Total Reagent Conc. Conc. reactions reactionvolume INVADER oligo mixture 1 0.05 10 1 10 (SEQ ID NO: 73, 74, and 75)(μM) Primary probe (SEQ ID 10 0.5 1 10 NO: 76) B1b3 (μM) FRET Cassette(SEQ ID 10 0.25 0.5 5 NO: 63) (μM) MOPS (mM) 400 10 0.5 5 CLEAVASE Xenzyme 50 2.5 1 10 (ng/μl) MgCl₂ (mM) 250 12.5 1 10 Total volume 5 50Aliquots of 5 μl of the master mix were added to each well. Reactionswere incubated at 62° C. for 4 hours and then read in the CYTOFLUOR asdescribed above.

The results are presented in FIG. 5 and demonstrate that the presence of1 μg of human genomic DNA does not exert a significant inhibitory effecton the INVADER assay designed to detect HPV 18 sequences.

Example 4 Simultaneous Detection of Multiple HPV Strains in a SinglePooled Reaction

In some situations, it may prove desirable to combine detection of manyHPV strains in a single reaction vessel. For example, it may be desiredto detect all high-risk HPV strains or all low-risk strains in a singlereaction mixture. In some cases, the output of a pooled reaction is aqualitative answer such as a positive result, indicating the presence ofone or more HPV strains, or a negative result, indicating the absence ofHPV.

Preferred oligonucleotide designs for pooling multiple INVADER reactionsin a single well may possess the following characteristics:

-   -   The oligonucleotides do not interact with one another to promote        excessive signal generation in the absence of a specific target.        Background in the INVADER assay may result from fragments of        certain oligonucleotides that are an intrinsic component of some        oligo synthesis mixtures. However, it is also possible for        groups of different oligonucleotides to assume structures that        are recognized and cleaved during the INVADER assay in the        absence of target.    -   The oligonucleotides do not interact with one another to        interfere significantly with signal generation in the presence        of a specific target.    -   Performance of a given oligonucleotide set is comparable when        tested in the pooled mixture and individually.

Pooled assays are created by combining probe and INVADERoligonucleotides in subcombinations and then assessing performance oneach target by comparing signal generation and FOZ of theoligonucleotides in the pool to detection of that target in reactionscontaining only the probe and INVADER oligonucleotides designed todetect it. In the event that a given oligonucleotide set is adverselyaffected by combination with other oligo sets in a single reactionvessel, e.g. generates excessive background or fails to generate theexpected levels of target-specific signal, in some cases it is possibleto swap in an alternative oligonucleotide set useful for determining thepresence of the same HPV strain. In other cases, it is possible tomerely choose a different 5′ arm for a particular probe oligonucleotideto reduce non-specific background generation or signal inhibition. Insome cases, it is possible to detect the alternative strand of aparticular target sequence, thereby altering the composition of theoligonucleotides and making them suitable for detection of the target ina pooled assay. In each case, the measure of a successfully performingassay is yield of statistically significant signal over background (FOZ)in the presence of the desired targets, e.g. ≧1.15 with t-test fromneighbor <0.05.

Ultimately, candidate oligonucleotide designs are pooled in variouscombinations and tested against a sample containing purified HPV DNA orpartial HPV sequences. Optimally, samples of all HPV strains beingtested are evaluated individually with the pooled oligonucleotide setsto confirm that target-specific signal is generated for each desiredstrain. Similarly, HPV negative samples or samples containing strains ofHPV not desired to be tested (e.g. low risk strains, HPV 1, or othernon-cervical strains) are also tested against the pools to confirm thatthey do not generate statistically significant FOZ.

Example 5 Detection of Multiple HPV Strains in Cervical Samples

The methods and compositions of the present invention were used, asdescribed in Example 4, to detect multiple strains of HPV in cervicalsamples.

INVADER Oligonucleotide Designs

The INVADER assay was designed to detect high-risk HPV strains including16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 67, 68, and 70. Probesets were combined into three pools based on HPV genetic phylogeny.Probe sets (e.g., probe and INVADER oligonucleotides) were designed tohybridize to at least 2 different target regions for each HPV strain toaccommodate for sequence polymorphisms and increase analyticalsensitivity (See, e.g., FIG. 6). Multiple HPV strains are detected byeach of the three pools using the INVADER assay (See, e.g., FIG. 6).Probe and INVADER oligonucleotide sequences are listed in FIG. 7. Theprobe and INVADER oligonucleotide sequences in FIG. 10 may also be used.An internal control assay (alpha-actin) is included in each pool tomeasure the relative amount of genomic DNA levels in the samples and toprovide a semi-quantitative method for HPV titer. The HPV specificprobes in the A7 and A9 pools contained arm 1 (CGCGCCGAGG; SEQ ID NOS:85, 88, 91, 94, 97, 101, 105, 108, 110, and 114) and utilized thecorresponding FAM FRET cassette(Fam-TCT-Z28-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG-hex, SEQ ID NO: 119). TheHPV specific probes in the A5/A6 pool contained arm AH9 (GGCAGTCTGGGAGT,SEQ ID NO: 77, 79, 81, and 83) and utilized the FAM FRET cassette(Fam-TCT-Z28-AGCCGGTTTTCCGGCTGAGAACTCCCAGACTGCC-hex, SEQ ID NO: 120).The alpha-actin assay contained arm 3(ACGGACGCGGAG; SEQ ID NO: 117) andutilized the RED FRET cassette(Red-TCT-Z28-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-hex, SEQ ID NO. 121).

INVADER Assay Reagents and Methods

Preparation of genomic DNA from cervical samples: DNA was isolated fromcervical samples obtained from a clinical laboratory using PUREGENE(Gentra Systems) DNA Purification Kit. The extraction procedure wasmodified to increase DNA yield and purity from this type of specimenusing the following procedure:

1. Remove 1 ml of cervical specimen and transfer to 1.5 ml tube.

2. Centrifuge cells at 16000 g for 5 min.

3. Remove supernatant and resuspend pellet in Cell Lysis Solution

4. Heat lysates at 99° C. for 10 minutes. Let cool to room temperature.

5. Add proteinase K and incubate at 55 for 1 hour.

6. Add 100 μl of protein precipitation solution.

7. Vortex samples vigorously for 20 seconds. Place on ice for 10minutes.

8. Centrifuge at 16000 g for 5 min.

9. Pour off the supernatant into a clean 1.5 ml tube containing 1.5 μlof glycogen (20 mg/ml)

10. Add 300 μl of 100% isopropanol.

11. Mix the sample gently by inverting 50 times.

12. Centrifuge at 16000 g for 5 min.

13. Pour off the supernatant and drain tube on clean absorbent paper.

14. Add 500 μl of 70% ethanol and invert the tube to wash the DNApellet.

15. Centrifuge at 16000 g for 2 min. Carefully pour off supernatant.

16. Invert and drain the tube on a clean absorbant paper and allow toair dry for 10-15 min.

17. Add 100 μl of distilled water to the pellet.

18. Let sit at room temperature overnight.

10 μl aliquots of each genomic DNA sample or no target control (10 ng/μltRNA) were added to a 96 well microtiter plate. Samples were overlaidwith 20 μl mineral oil, denatured at 99° C. for 10 minutes and thencooled to 63° C. A 100 aliquot of the INVADER reaction mix was thenadded to each well and mixed by pipetting. An example of what iscontained in the INVADER assay reaction mix is shown below.

Amount Final per concentrations Component reaction (in 20 μl reaction)MgCl2 (70 mM) 4 μl 14 mM MgCl₂ HPV Pooled Primary probes 4 μl 0.5 μM ofeach probe and INVADER oligos/FAM FRET/MOPS 0.05 μM of each Invaderoligo Stock conc. 0.25 μM FRET cassette 2.5 μM of each probe 10 mM MOPS0.25 μM of each Invader oligo 1.25 μM of FRET cassette 40 mM MOPS AlphaActin Primary probe/ 1 μl 0.25 μM probe INVADER oligo/ RED FRET/MOPSStock conc. 0.05 μM Invader oligo 5 μM probe 0.25 μM FRET cassette 1 μMInvader oligo 10 mM MOPS 5 μM of FRET 40 mM MOPS CLEAVASE X enzyme 1 μl2 ng/μl (40 ng/μl) in CLEAVASE dilution bufferINVADER Assay Reactions

Reactions were incubated at 63° C. for 4 hours and then cooled to 4° C.prior to scanning in a CYTOFLUOR 4000 fluorescence plate reader (AppliedBiosystems, Foster City, Calif.). The settings used were: 485/20 nmexcitation/bandwidth and 530/25 nm emission/bandwidth for FAM dyedetection, and 560/20 nm excitation/bandwidth and 620/40 nmemission/bandwidth for RED dye detection. The instrument gain was setfor each dye so that the No Target Blank produced between 100-250Relative Fluorescence Units (RFUs). Microplates were also read in theGenios FL Plate reader (Tecan, Research Triangle Park, N.C.). Thesettings used were: 485/535 nm excitation/emission for FAM dyedetection, and 560/612 nm excitation/emission for RED dye detection. Theinstrument gain was set for each dye so that the No Target Blankproduced between 1000-2000 Relative Fluorescence Units (RFUs). Becausethe optimal gain setting can vary between instruments, the gain wasadjusted to provide the best signal/background ratio (e.g., sample rawsignal divided by the No Target Control signal) or No Target Controlsample readings. For directly reading the microplates, the probe heightof the microplate reader and the positioning of the plate was adjustedaccording to the manufacturer's recommendations.

The fluorescent signal from the Fam dye and the Red dye for the samplesand No Target Control (NTC) was used to calculate fold over zero (FOZ)values as shown below.FOZ_(Fam)dye=(RawSignal_(Fam)/NTC_(Fam))FOZ_(Red)=(RawSignal_(Red)/NTC_(Red))The Fam FOZ corresponds to the signal from the HPV assays, and the REDFOZ corresponds to the alpha-actin signal (See, e.g., FIG. 9).Results of INVADER Assays for Detection HPV in Cervical Specimens

Quantification of DNA concentration in cervical samples may be achievedusing various methods. For example, DNA concentration can be measuredusing the OliGreen ssDNA Quantitation kit (Molecular probes) or thealpha-actin INVADER assay (See, e.g., FIG. 8). To determine the amountof DNA present in each sample using the INVADER Assay, a control genomicDNA sample was serially diluted to generate a standard curve. Thealpha-actin INVADER assay standard curve was used to determine theamount of DNA present in each sample using a linear regression analysis.Both methods are useful for determining concentrations of DNA incervical samples. Since the signal from the alpha-actin INVADER assaycan be detected in the same well as the HPV INVADER assays, a separatequantitation step by OliGreen or measuring absorbance at 260 nm is notrequired.

The INVADER assay was used to detect the presence or absence of HPV(e.g., high-risk HPV strains) in cervical samples (See, e.g., FIG. 9).Each sample was tested in three separate wells of a microtiter platecontaining either the A5/A6, A7 or A9 INVADER reaction mix. All wellscontained the alpha-actin oligonucleotides and FRET cassette. Sampleswere considered to be HPV positive if the FAM FOZ values were greaterthan 3, HPV negative if the FAM FOZ values were less than 2, andequivocal if the FAM FOZ values were between 2 and 3. Of the 45 cervicalsamples tested, there were 21 positive samples, 23 negative samples, and1 equivocal sample. Four of the samples were determined, using themethods of the present invention, to be co-infected with multiple HPVstrains (See, e.g., FIG. 9, samples 10-658, 10-662, 10-677 and 10-682).

All publications and patents mentioned in the above specification areherein incorporated by reference as if expressly set forth herein.Various modifications and variations of the described assays of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention that are obvious tothose skilled in relevant fields are intended to be within the scope ofthe following claims.

We claim:
 1. A method for detecting the presence or absence of a HPVtarget nucleic acid in a sample, wherein said HPV target nucleic acid isfrom one or more strains in a set of strains of HPV, comprising: a)providing: i) a sample suspected of containing a HPV target nucleicacid; ii) a composition comprising a mixture of differentoligonucleotide probe sets, wherein: A) each of said oligonucleotideprobe sets comprises a first oligonucleotide and a secondoligonucleotide that in combination form an invasive cleavage structurewith target nucleic acid from at least two different strains of HPV insaid set of strains of HPV; B) each of said oligonucleotide probe setsforms an invasive cleavage structure with target nucleic acid from fewerthan all of the different strains in said set of strains of HPV; and C)at least two of said probe sets in said mixture are configured to forman invasive cleavage structure with target nucleic acid from eachdifferent strain of HPV in said set of strains of HPV; and iii) a 5′nuclease; b) exposing said sample to said composition under reactionconditions in which, if HPV target nucleic acid from one or more strainsin said set of strains is present, at least two of said oligonucleotideprobe sets hybridize to said HPV target nucleic acid to form at leasttwo invasive cleavage structures, and in which said at least twoinvasive cleavage structures are cleaved with said 5′ nuclease; and c)detecting cleavage of said invasive cleavage structures, whereindetection of cleavage of said invasive cleavage structures is indicativeof the presence of HPV target nucleic acid from at least one strain ofsaid set of strains of HPV in said sample.
 2. The method of claim 1,wherein said detection of cleavage is indicative of the presence of HPVtarget nucleic acid from at least one strain from the group of strainsconsisting of strains 16, 16Ty2, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,58iso, 59, 66, 67, 68, 68var, 69, 70, and
 82. 3. The method of claim 1,wherein said mixture detects HPV strains 51, 56, and
 66. 4. The methodof claim 1, wherein said mixture detects HPV strains 39, 68, 68var, 45,59 and
 18. 5. The method of claim 1, wherein said mixture detects HPVstrains 33, 52, 58, 31, 35 and
 16. 6. The method of claim 1, whereinsaid mixture comprises the group of oligonucleotides consisting of SEQID NOS. 47, 51, 52, 79, and
 80. 7. The method of claim 1, wherein saidmixture comprises the group of oligonucleotides consisting of SEQ IDNOS. 37, 38, 94, 95, 127, 129 and
 142. 8. The method of claim 1, whereinsaid mixture comprises the group of oligonucleotides consisting of SEQID NOS. 12, 13, 37, 38, 47, 51, 52, 79-80, 91, 94, 127, 129, and
 142. 9.The method of claim 1, wherein said 5′ nuclease is a FEN-1 endonuclease.10. The method of claim 1, wherein said 5′ nuclease is thermostable. 11.The method of claim 1, wherein said HPV target nucleic acid is amplifiedprior to said exposure step.
 12. The method of claim 1, wherein saidmixture of oligonucleotide probe sets further comprises a pair ofoligonucleotide primers configured to amplify a sequence contained insaid HPV target nucleic acid in a polymerase chain reaction.
 13. Themethod of claim 1, wherein said HPV target nucleic acid is a syntheticnucleic acid.
 14. The method of claim 1, wherein said HPV target nucleicacid is genomic HPV nucleic acid.
 15. The method of claim 1, wherein twoor more mixtures of oligonucleotide probes are used together to detectsaid HPV target nucleic acid.
 16. The method of claim 1, wherein two ormore of said mixtures of oligonucleotide probes are used together todetect all high-risk HPV strains.
 17. The method of claim 1, whereinsaid sample is from the group consisting of cervical cells, cervicalsecretions, epithelial cells, respiratory secretions, urethral cells,cells of the anogenital region, urine, saliva and biopsy tissue.
 18. Themethod of claim 1, wherein said second oligonucleotides comprise adetectable label.
 19. The method of claim 1, wherein cleavage of saidinvasive cleavage structures cleaves said second oligonucleotide toproduce a cleaved 5′ region of said second oligonucleotide, wherein saidproviding step further comprises providing an oligonucleotide FRETcassette, wherein said oligonucleotide FRET cassette comprises a set ofinteractive labels, wherein said oligonucleotide FRET cassette isconfigured to form a second invasive cleavage structure with saidcleaved 5′ region of said second oligonucleotide.
 20. The method ofclaim 19, wherein said cleaving further comprises cleaving said secondinvasive cleavage structure.
 21. The method of claim 19, wherein saiddetecting comprises detecting and/or measuring a signal produced fromone or more members of a set of interactive labels.